Polymer coating of cells

ABSTRACT

Polymers and methods of making them are described. The polymers may be used to coat living cells. The polymer-coated cells are useful in cell therapy applications.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.60/662,612 and 60/662,617, both filed Mar. 16, 2005, both of which arehereby incorporated by reference in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates generally to coating and delivery of cells. Thecoating material includes biodegradable polymers containing hydrophobicgroups, hydrophilic groups, and reporters. The polymer coated cells maybe used in cell-based therapies.

2. Description of the Related Art

While therapeutic advances in the last decade have produced manyeffective drugs and treatments, such treatments usually are unable tocompletely correct or reverse disease states because most disease statesare caused by complex interactions between cell components. Cell therapyholds much promise to not only provide relief from disease symptoms, butto actually reverse disease states. Blood transfusions and bone marrowtransplants are examples of successful use of cell-based therapeutics

There are currently no effective therapies for many acquired andcongenital cardiovascular, pulmonary, and hematologic diseases anddisorders. Recent discoveries in stem cell biology present newopportunities for the use of cell-based therapies in disease areas withcritical, unmet medical needs. Adult, cord blood, embryonic and fetalstem cells hold great potential for use in new strategies aimed at theregeneration and repair of damaged or diseased cardiovascular, lung, andblood tissues.

SUMMARY OF THE INVENTION

Embodiments of the invention are directed to polymers which include atleast one recurring unit represented by a formula selected from formulas(I), (II), and (III):

wherein n is 1 or 2; wherein x and y are each individually integers offrom about 1 to about 500; wherein Z is an optional linker groupincluding from about zero to about 20 carbon atoms, from about zero toabout 5 oxygen atoms, from about zero to about five nitrogen atoms, fromabout zero to about 5 sulfur atoms, and from about zero to about fivephosphorous atoms; and wherein each W is individually biotin, a fattyacid, a fluorescent dye, an antibody, a peptide, a targeting ligand, apolysaccharide, or a negatively charged group. In preferred embodiments,the fatty acid includes a moiety which is oleic, stearyl, palmitic,linoleic, linolenoic, or cholesteryl. In preferred embodiments, thenegatively charged group is C(═O)O⁻, SO₃ ⁻, or PO₄ ²⁻.

In preferred embodiments, the polymer includes at least one recurringunit selected from a recurring unit of the formula (IV), a recurringunit of the formula (V), and a recurring unit of the formula (VI):

In some preferred embodiments, the polymer includes a recurring unit ofthe formula (IV). In some preferred embodiments, the polymer includes arecurring unit of the formula (V). In some preferred embodiments, thepolymer includes a recurring unit of the formula (VI). In some preferredembodiments, the polymer includes a recurring unit of the formula (IV),a recurring unit of the formula (V), and a recurring unit of the formula(VI).

Preferred embodiments are directed to a polymer which includes arecurring unit of the formula (VII):

In preferred embodiments, the polymer includes a recurring unit of theformula (VIII):

In preferred embodiments, the polymer includes a recurring unit of theformula (IX):

Embodiments of the invention are directed to methods of making thepolymers that include a recurring unit of the formula (I) as describedabove, which includes amidating a polymer comprising a recurring unit ofthe formula (VII) as described above with a reagent which isNHS-dPEG4-Biotin, NHS-dPEG4-peptide, glutaric anhydride, fatty acidchloride, or activated ester NHS.

Embodiments of the invention are directed to methods of making polymerswhich include recurring units of the formula (II) as described above,which include amidating a polymer that includes a recurring unitselected from a glutamic acid recurring unit and a glutamic acid saltrecurring unit, with an amino-peptide and a fatty acid amine.

Embodiments of the invention are directed to methods of making polymerswhich include a recurring unit of the formula (III) as described above,which include amidating a polymer that includes a recurring unitselected from a lysine recurring unit and a lysine salt recurring unit,with a reagent which is NHS-dPEG4-Biotin, NHS-dPEG4-peptide, glutaricanhydride, fatty acid chloride, or activated ester NHS.

Embodiments of the invention are directed to methods of making polymersthat include a recurring unit of the formula (IV) as described above,which includes amidating a polymer that includes a recurring unit of theformula (VII) as described above with oleic chloride.

Embodiments of the invention are directed to methods of making polymersthat include a recurring unit of the formula (V) as described above,which include amidating a polymer that includes a recurring unit of theformula (VII) as described above with glutaric anhydride and potassiumcarbonate.

Embodiments of the invention are directed to methods of making polymersthat include a recurring unit of the formula (VI) as described above,which include amidating a polymer that includes a recurring unit of theformula (VII) as described above with NHS-dPEG4-Biotin.

Embodiments of the invention are directed to methods of making polymersthat include a recurring unit of the formula (VII) as described above,which include removing a protecting group from a polymer that includes arecurring unit of the formula (VIII) as described above viapalladium/carbon catalytic hydrogenation.

Embodiments of the invention are directed to methods of making a polymerwhich includes a recurring unit of the formula (VII) as described above,which include treating a polymer that includes a recurring unit of theformula (IX) as described above with 20% piperidine.

Embodiments of the invention are directed to methods of making polymerswhich include a recurring unit of the formula (VIII) as described above,which include amidating a diamine and a glutamic amino acid derivative,wherein the diamine is represented by the formula:

and wherein the glutamic amino acid derivative is represented by theformula:

Embodiments of the invention are directed to methods of making polymersthat include a recurring unit of the formula (IX) as described above,which include amidating a diamine and a glutamic amino acid derivative,wherein the diamine is represented by the formula:

and wherein the glutamic amino acid derivative is represented by theformula:

Embodiments of the invention are directed to a polymer-coated cell whichincludes a living cell and a polymer which includes at least onerecurring unit represented by a formula selected from formulas (I),(II), and (III) as described above, where the polymer is non-covalentlyattached to at least a portion of the exterior of the living cell.Preferably, the living cell is an epithelial cell, an endothelial cell,a progenitor cell, a mature stem cell, or an embryonic stem cell.

In preferred polymer-coated cell embodiments, the polymer includes atleast one recurring unit selected from a recurring unit of the formula(IV), a recurring unit of the formula (V), and a recurring unit of theformula (VI) as described above.

In preferred embodiments, W in formulas (I), (II), and (III) is atargeting ligand. More preferably, the targeting ligand is transferrinor epidermal growth factor. In some preferred embodiments, the antibodyis to a receptor that is overexpressed in cancer cells. In preferredembodiments, the fatty acid includes a moiety which is oleic, stearyl,palmitic, linoleic, linolenoic, or cholesteryl. In preferredembodiments, the negatively charged group is C(═O)O⁻, SO₃ ⁻, or PO₄ ²⁻.

In preferred polymer-coated cell embodiments, the living cell is amammalian cell. More preferably, the living cell is a human cell.

Embodiments of the invention are directed to a method for coating aliving cell, comprising intermixing the living cell with a polymer whichincludes at least one recurring unit represented by a formula selectedfrom formulas (I), (II), and (III) as described above, wherein thepolymer is intermixed with the living cell in an amount effective to atleast partially coat the exterior of the living cell.

In preferred embodiments, the polymer includes at least one recurringunit selected from a recurring unit of the formula (IV), a recurringunit of the formula (V), and a recurring unit of the formula (VI), asdescribed above.

In preferred polymer-coated cell embodiments, the living cell is anepithelial cell, an endothelial cell, a progenitor cell, a mature stemcell, or an embryonic stem cell. In preferred embodiments, the fattyacid includes a moiety which is oleic, stearyl, palmitic, linoleic,linolenoic, and cholesteryl. In preferred embodiments, the negativelycharged group is C(═O)O⁻, SO₃ ⁻, or PO₄ ²⁻.

In preferred embodiments, the living cell is a mammalian cell. Morepreferably, the living cell is a human cell.

Embodiments of the inventions are directed to a method of treating adisease or injury which comprises administering the polymer coated cellto an individual in need thereof. In preferred embodiment, theindividual is a mammal. Preferably, the mammal is a human.

In preferred embodiments, the administration is by injection.Preferably, the administration is by injection into an organ which isdiseased or injured or into a vascular system which feeds the diseasedor injured organ. In some preferred embodiments, administration is intoa tail vein of a mouse.

Further aspects, features and advantages of this invention will becomeapparent from the detailed description of the preferred embodimentswhich follow.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other feature of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

FIG. 1 shows a reaction scheme according to an embodiment forpreparation of polymers by amidation of a diamine and a glutamic aminoacid derivative.

FIG. 2 shows a reaction scheme according to an embodiment forpreparation of polymers by amidation of the polymer 6 withNHS-dPEG₄-Biotin, glutaric anhydride, and oleic chloride.

FIG. 3 shows the polymer IA produced by the reaction scheme of FIG. 2 asdescribed in Example 6.

FIG. 4 shows lung cancer cells in the presence (left side) and absence(right side) of Polymer IA. Both treatments were treated withStreptavidin-Alexa 448 to visualize the binding.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

These and other features of this invention will now be described withreference to the drawings of preferred embodiments which are intended toillustrate and not to limit the invention.

The invention relates generally to biodegradable polymer, methods formaking them, and methods of using them for various applications,including cell delivery and transplantation, and particularly fortransplantation of autologous and non-autologous progenitor cells orstem cells. In an embodiment, a method provides therapeutic effects fora wide range of diseases in which cell-based products have been reducedor lost due to disease or injury. These cell-based products may bereplaced by cell implantation techniques.

The term “progenitor cells” is a broad term and refers to cells whichare capable of development into a distinct cell types or lineages withina tissue or organ by a series of cell divisions.

The term “stem cell” is a broad term and refers to cells which arecapable of regeneration into multiple cell types or lineages. The term“stem cell” includes both adult stem cells and embryonic stem cells.

Polymers and Methods for Making Them

An embodiment provides a polymer comprising at least one recurring unitrepresented by a formula selected from the group consisting of formulas(I), (II), and (III):

wherein n is 1 or 2; wherein x and y are each individually integers offrom about 1 to about 500; wherein Z is an optional linker groupcomprising from about zero to about 20 carbon atoms, from about zero toabout 5 oxygen atoms, from about zero to about five nitrogen atoms, fromabout zero to about 5 sulfur atoms, and from about zero to about fivephosphorous atoms; and wherein each W is individually selected from thegroups consisting of biotin, a fatty acid, a fluorescent dye, anantibody, a peptide, a targeting ligand, a polysaccharide, and anegatively charged group. In an embodiment, the fatty acid comprises amoiety selected from the group consisting of oleic, stearyl, palmitic,linoleic, linolenoic, and cholesteryl. In an embodiment, the negativelycharged group is selected from the group consisting of C(═O)O⁻, SO₃ ⁻,or PO₄ ²⁻.

Examples of polymers that comprise recurring units of the formula (I)include polymers that comprise recurring units of the formula (IV),polymers that comprise recurring units of the formula (V), and polymersthat comprise recurring units of the formula (VI):

Thus, in an embodiment, a polymer that comprises a recurring unit of theformula (I) comprises at least one recurring unit selected from thegroup consisting of a recurring unit of the formula (IV), a recurringunit of the formula (V), and a recurring unit of the formula (VI).Polymers that comprise a recurring unit of the formula (I) may becopolymers. For example, in an embodiment, a polymer that comprises arecurring unit of the formula (I) comprises two or more recurring unitsselected from the group consisting of a recurring unit of the formula(IV), a recurring unit of the formula (V), and a recurring unit of theformula (VI). As another example, a polymer that comprises a recurringunit of the formula (I) comprises a recurring unit of the formula (IV),a recurring unit of the formula (V), and a recurring unit of the formula(VI).

Polymers that comprise a recurring unit of the formula (I) may beprepared in various ways. For example, an embodiment provides a polymerthat comprises a recurring unit of the formula (VII):

Polymers that comprise a recurring unit of the formula (I) may beprepared by a process that comprises amidating a polymer that comprisesa recurring unit of the formula (VII) with a reagent selected from thegroup consisting of NHS-dPEG₄-Biotin, NHS-dPEG₄-peptide, glutaricanhydride, fatty acid chloride, and activated ester NHS. For example, inone embodiment, a method of making a polymer that comprises a recurringunit of the formula (IV) comprises amidating a polymer that comprises arecurring unit of the formula (VII) with oleic chloride. In anotherembodiment, a method of making a polymer that comprises a recurring unitof the formula (V) comprises amidating a polymer that comprises arecurring unit of the formula (VII) with glutaric anhydride andpotassium carbonate. In yet another embodiment, a method of making apolymer that comprises a recurring unit of the formula (VI) comprisesamidating a polymer that comprises a recurring unit of the formula (VII)with NHS-dPEG₄-Biotin. The working examples provided below describespecific reaction conditions for carrying out an exemplary amidationreaction. Routine experimentation may be used to identify appropriateamidation conditions to prepare other polymers that comprise a recurringunit of the formula (I).

Polymers that comprise a recurring unit of the formula (II) may beprepared by a process that comprises amidating a polymer that comprisesa recurring unit selected from the group consisting of glutamic acidrecurring unit and glutamic acid salt recurring unit, with anamino-peptide and a fatty acid amine. Polymers that comprise a recurringunit of the formula (III) may be prepared by a process that comprisesamidating a polymer that comprises a recurring unit selected from thegroup consisting of lysine recurring unit and lysine salt recurringunit, with a reagent selected from the group consisting ofNHS-dPEG₄-Biotin, NHS-dPEG₄-peptide, glutaric anhydride, fatty acidchloride, and activated ester NHS. Routine experimentation may be usedto identify appropriate amidation conditions to prepare polymers thatcomprise recurring units of the formula II and/or III.

Polymers that comprise a recurring unit of the formula (VII) are anotherembodiment. Such polymers are useful for, e.g., preparing polymers thatcomprise a recurring unit of the formula (I). Polymers that comprise arecurring unit of the formula (VII) may be prepared by a process thatcomprises removing a protecting group from a polymer that comprises arecurring unit of the formula (VIII) via palladium/carbon catalytichydrogenation:

Polymers that comprise a recurring unit of the formula (VII) may also beprepared by a process that comprises treating a polymer that comprises arecurring unit of the formula (IX) with 20% piperidine:

Polymers that comprise a recurring unit of the formula (VIII) andpolymers that comprise a recurring unit of the formula (IX) are alsoembodiments. Such polymers are useful for, e.g., preparing polymers thatcomprise a recurring unit of the formula (VII). Polymers that comprise arecurring unit of the formula (VIII) may be prepared by a process thatcomprises amidating a diamine and a glutamic amino acid derivative,where the diamine is represented by the formula:

and where the glutamic amino acid derivative is represented by theformula:

Likewise, polymers that comprise a recurring unit of the formula (IX)may also be prepared by a process that comprises amidating a diamine anda glutamic amino acid derivative, where the diamine is represented bythe formula:

and where the glutamic amino acid derivative is represented by theformula:

Polymer-Coated Cells and Methods for Making and Using Them

A variety of disease states may be treated according to the embodiments.These include neurologic diseases (e.g. Parkinson's disease, MultipleSclerosis), cardiovascular disease (myocardial ischemia, repair andregeneration of infarcted myocardium, hepatic (liver failure), diabetes,skin, and renal failure (chronic renal failure, acute renal failure).

The target tissue may be an organ such as heart, brain, kidney, skin,liver, muscle, spleen, lung, spinal cord and bone marrow. The individualbeing treated is preferably a mammal. The embodiments may be applied inparticular to humans and veterinary animals which include but are notlimited to horses, sheep, cows, dogs, and cats. Administration may be byinjection directly into the organ in need of treatment or by injectioninto the vascular system feeding the organ in need of treatment.

There are four basic issues for cell-based therapies. These aremobilization of the cells, homing to the target site, integration intothe native tissue or organ and survival of the implanted cells. In thecontext of the invention, the terms “targeting” and “homing” are usedinterchangeably. The polymer coatings according to the presentembodiments are directed particularly to problems relating tointegration into native tissue and survival of implanted cells. Bycoating of the cells with the polymers according to the embodiments, thecells may be protected in the blood for several hours. The polymercoated cells are protected from the immune response of the host. Thesecoatings protect the cell therapeutic while allowing passage of vitalnutrients including oxygen. Note that the polymer does not need tocompletely coat the cell surface in order to provide protection.

The selection of cell type is a function of the disease which is beingtreated. For example, skeletal myocytes would be injected intopost-myocardial infarction scar tissue; neuronal cells would beadministered to the brain of patients with Parkinson's Disease. Cellsources which may be used include embryonic stem (ES) cells, adult stemcells, progenitor cells such as skeletal myoblasts, fetal and neonatalcariomyocytes, and chord blood.

The derivation of human ES cells has opened new avenues for using thesecells for cellular therapies. ES cells are thought to be a trulypluripotent stem cell capable of self-renewal, can differentiate intoall three germ cell layers, and have been shown to form hematopoieticcells and cardiomyocytes. Cells with these properties hold the promiseof being able to repair or replace cells or tissues that are damaged ordestroyed by many of the most devastating diseases and disabilities. Oneof the current advantages of using ES cells, as compared to adult stemcells, is that ES cells will proliferate in vitro, and have been used togenerate a broad range of cell types through directed differentiation.

Adult stem cells also offer great promise as cell-based therapies andare free of the ethical issues surrounding ES cells. Hematopoietic stemcells give rise to all blood cells and have been used to treat seriousblood disorders, malignant disease, and inherited diseases. It has beendemonstrated that hematopoietic stem cells can differentiate intocardiac muscle cells, vascular cells, lung epithelium, neural cells,glial cells and other cell lineages. Recently the ability oftransplanted or mobilized hematopoietic stem cells to engraft and repairheart muscle and vascular tissue damaged by ischemia has beendemonstrated in animal models. In addition, bone marrow derived cellshave been demonstrated to engraft as alveolar type 1 epithelial cellsand as bronchial epithelial cells.

Cardiovascular and lung tissues may also contain progenitor or stemcells that under the correct conditions could be induced to proliferateand repair cellular damage. For instance, recent findings suggest that asub-population of fetal proliferative alveolar epithelial stem cells ispresent in adult lung. In addition, other tissues such as skin, liver,brain, and muscle have progenitor or stem cell populations that mayprovide additional sources of cells for cellular therapies.

For neovascularization of ischemic myocardium, endothelial progenitorcells may be injected to the target area to promote new vessel growth.The cells are isolated from the mononuclear cell fraction of bone marrowor peripheral blood. The cells may be whole isolated cells or the cellsmay first be expanded in culture. Other examples include treatment ofskin disease with replacement grafts. Skeletal stem cell implantationmay be used for bone regeneration. Chondrocytes may be used to repairjoint cartilage. Acute and chronic renal failure may be treated withstem/progenitor cells.

The cell source may be either an autologous source or a non-autologoussource. In some embodiments, the cells may be genetically modified. Incases where an adequate supply of cells is not possible from the patientdue to the disease or other condition, non-autologous sources may beused. Non-autologous cells include allogeneic and xenogeneic cells.Non-autologous sources must overcome the natural host immunologicrejection processes. The polymer coating according to the embodimentsprovides protection from the host immune response.

The use of autologous cells generally involves obtaining the patient'sown cells, expanding the cells in vitro in large quantities over severalweeks, and reintroducing the cells in a site-specific manner.

A variety of means for administering cells to individuals in need oftreatment will be apparent to those of skill in the art. Such methodsinclude injection of the cells into a target site in a subject. Cellsmay be inserted into a delivery device which facilitates introduction byinjection or implantation into the subjects. Such delivery devices mayinclude tubes, e.g., catheters, for injecting cells and fluids into thebody of a recipient subject. In a preferred embodiment, the tubesadditionally have a needle, e.g., a syringe, through which the cells ofthe embodiments can be introduced into the subject at a desiredlocation. In a preferred embodiment, cells are formulated foradministration into a blood vessel via a catheter (where the term“catheter” is intended to include any of the various tube-like systemsfor delivery of substances to a blood vessel). The cells may be preparedfor delivery in a variety of different forms. For example, the cells maybe suspended in a solution or gel. Cells may be mixed with apharmaceutically acceptable carrier or diluent in which the cells of theembodiments remain viable. Pharmaceutically acceptable carriers anddiluents include saline, aqueous buffer solutions, solvents and/ordispersion media. The use of such carriers and diluents is well known inthe art. The solution is preferably sterile and fluid, and will often beisotonic. Preferably, the solution is stable under the conditions ofmanufacture and storage and preserved against the contaminating actionof microorganisms such as bacteria and fungi through the use of, forexample, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, andthe like.

Modes of administration of the polymer coated cells include but are notlimited to systemic intracardiac, intracoronary, intravenous orintra-arterial injection and injection directly into the tissue at theintended site of activity. The preparation can be administered by anyconvenient route, for example by infusion or bolus injection and can beadministered together with other biologically active agents.Administration is preferably systemic. Most preferably, the site ofadministration is close to or nearest the intended site of activity. Insome embodiments, the polymer coated cells will migrate or home to thetissue or organ in need of treatment in response to chemotactic factorsproduced due to the injury without specific modification of the polymercoated cells for targeting.

Modifications of the polymer coating provide for homing of the cells tothe target site. Protein targeting agents such as antibodies or proteinsthat bind to specific membrane sites may be used to target the polymercoated cells to the target organ or tissue. In some embodiments of themethods described herein, the polymer coated cells are modified prior toimplantation into the individual so as to promote their targeting totissue or organ in need of treatment. For example, the polymer mayinclude an antibody which binds an antigen that is abundant at thetarget site, that is, at the site of the tissue or organ which isdiseased or in need of treatment.

For example, monoclonal antibodies are known that specifically targetcancer cells. Many of these are antibodies to growth factor receptorswhich are preferentially expressed on the surface of cancer cells. Theseinclude the humanized monoclonal antibody trastuzumab (Herceptin) whichtargets the HER-2/neu oncogene (Sato, et al. (2005) Int. J. RadiationOncology Biol. Phys. vol. 61 (1): 203-211). The HER-2/neu oncogene isfound in ovarian cancer, lung cancer, gastric cancer, oral squamous cellcarcinoma, breast cancer, and esophageal cancer. BLCA-38 monoclonalantibody has been shown to target prostate and bladder cancer (Russell,et al. (2004) Cancer Immunol Immunother. vol. 53:995-1004). Othermonoclonal antibodies are known and it is within the level of skill inthe art to select a monoclonal antibody appropriate to the cancer orother disease or injury to be treated.

Migration of polymer coated cells to target tissues may be enhanced bygenetic modification, e.g., introduction of an exogenous nucleic acidencoding a homing molecule into the cells. Examples of homing moleculesinclude receptors specific to the target tissue such as chemokinereceptors, interleukin receptors, estrogen receptors, and integrinreceptors.

In preferred embodiments, a receptor ligand such as transferrin orepidermal growth factor is included in the polymer for homing to cancercells. These ligands provide specific targeting to receptors on tumorcells. Thus, delivery of the coated cells is localized to the area inneed of treatment for maximum effectiveness. Also any adverse effects ofthe treatment are localized to minimize unwanted side effects.

Another method of homing a cell such as a stem cell to an injured tissueis carried out by increasing the amount of an injury-associatedpolypeptide, e.g., a cytokine or adhesion protein, in the injuredtissue. The method increases the number of stem cells in an area ofinjured tissue compared to the number of stem cells in the area in theabsence of an exogenous injury-associated polypeptide or nucleic acidencoding such a polypeptide. For example, identification ofinjury-associated polypeptides, e.g., growth factors, activateendogenous mechanisms of repair in the heart such as proliferation anddifferentiation of cardiac progenitor cells. The injured tissue iscontacted with a nucleic acid encoding a protein such as a cytokine oradhesion protein. Alternatively, cells such as fibroblast cellsexpressing exogenous nucleic acid molecules encoding the cytokine oradhesion protein are introduced to the site of injury.

In one embodiment, the cells optionally contain an exogenous nucleicacid encoding a gene product, which increases endocrine action of thecell, e.g., a gene encoding a hormone, or a paracrine action of thecell. For example, stem cells are genetically modified to contain anexogenous nucleic acid encoding a bone morphogenetic factor andengrafted into bone, cartilage, or tooth tissue, e.g., to treatperiodontitis.

The cells optionally also include nucleic acids encoding otherbiologically active or therapeutic proteins or polypeptides, e.g.,angiogenic factors, extracellular matrix proteins, cytokines or growthfactors. For example, cells to be engrafted into pancreatic tissuecontain a nucleic acid(s) encoding insulin or insulin precursormolecules. The cells also optionally include nucleic acids encoding geneproducts that decrease transplant rejection, e.g., CTLA4Ig CD40 ligand,or decrease development of transplant arteriosclerosis, e.g., induciblenitric oxide synthase (iNOS).

Tissue specificity is a fundamental problem for gene therapy as proteinsthat are therapeutic in target cells also may be harmful to normaltissue. Thus non cell-specific expression of a transgene has thepotential for inducing metabolic and physiologic mechanisms that couldresult in pathology over the long term. Localized injections can providecertain degree of localized expression of the targeting vector, however,there may still be a spill over into the circulation which will affectother cells and organs. In some embodiments, transcriptionally targetedvectors may be used that can restrict the expression of the therapeuticproteins primarily to the target cells by the use of tissue-specificpromoters.

Once the cells are implanted, maintenance of the cells is dependent uponadequate nutrient and oxygen delivery to the implanted cells. Thepolymer cell coating according to the embodiments allows for entry ofoxygen and other nutrients into the coated cell.

If administration of an immunosuppressant is indicated, one skilled inthe art would be able to select a suitable immunosuppressant agent suchas cyclosporine, sirolimus, rapamycin, or tacrolimus.

In some embodiments, the polymer includes a label. The label may be adye or fluorescent label such as Alexa Fluor dyes, BODIPY dyes, Cascadeblue dyes, coumarin, Digoxigenin, Environment-Sensitive dyes,Fluorescein, FITC, Haptens, Lissamine Rhodamine B dyes, NBD, OregonGreen dyes, Blue-Fluorescent dyes, photosensitizers, QSY Fluorescent-Dyequenchers, Rhodamine 6G dyes, Rhodamine green dyes, Rhodamine red dyes,tetramethylrhodamine, or Texas red dyes.

Alternatively, the polymer may include a radioactive or a radio-opaquedye such as PET isotopes (18F, 124I, or 76Br) or a radio-opaque dye,e.g., an iodine compound, barium or barium sulfate or gastrografin andthe like. After the polymer has bound to the cell, it may then bevisualized using well known techniques such as PET, MRI, CT, SPECT, etc(see Molecular Imaging of Gene Expression and Protein Function In VivoWith PET and SPECT, Vijay Sharma, PhD, Gary D. Luker, MD, and DavidPiwnica-Worms, MD, Ph.D., JOURNAL OF MAGNETIC RESONANCE IMAGING16:336-351 (2002)).

In preferred embodiments, the polymers described herein may be usedadvantageously as a coating for cells, particularly cells for use incell-based therapy applications as described above.

In preferred embodiments, solutions are prepared containing polymers,prepared as described above. Generally, these solutions are prepared ata concentration of 1-50 μg/ml, more preferably about 5-10 μg/ml. Thepolymer may include any of the modifications described above, includingbut not limited to cell targeting agents, intracellular-targetingmoiety, cytotoxicity reductive reagents, cell binding reagents, cellgrowing reagents, cell stimulating reagents, cell inhibiting reagents,compounds for culturing specific cells, and/or neutral polymers such aspolyethylene glycol co-polymers. In preferred embodiments, a probe isadded to the polymer solution.

Cells are seeded in an appropriate vessel such as a multiwell plate,petri dish, or test tube and allowed to grow until 20-70% confluency,preferably 40-60% confluency, most preferably about 50% confluency underconditions appropriate for the cell type used. Any cell type may becoated with the polymers as described herein, including both eukaryoticand prokaryotic cell types. Preferably, the cell is a eukaryotic cell,more preferably the cell type is a mammalian cell. Most preferred celltypes include embryonic stem cells, adult stem cells, skeletalmyoblasts, fetal and neonatal cariomyocytes and chord blood cells. Thecells are checked to insure viability using criteria well known in theart including evaluation of cell morphology and cytotoxic assays. Insome preferred embodiments, the buffer/cell culturing solution may beremoved and the cells may be washed with a physiologically compatiblebuffer such as PBS. However, any physiological buffer may be usedincluding but not limited to bicarbonate buffers, monobasic phosphate,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),4-morpholinepropanesulfonic acid (MOPS),1,4-piperazinebis(ethanesulfonic acid) (PIPES),N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES),tris(hydroxymethyl)aminomethane (TRIS BASE), andtris(hydroxymethyl)aminomethane hydrochloride (TRIS.HCl). The polymersolution is then added to the cells on the vessel. The cells areincubated for an appropriate period with the polymer solution to providea coated cell. Convenient incubation times may be from 5 min. to 2hours, preferably 10 min. to 30 min., most preferably about 15 min. Thecells may optionally be washed with physiological buffer as describedabove.

Alternatively, cells may be harvested by centrifugation. The cells arepreferably dividing cells. The harvested cells may optionally be washedin an appropriate buffer as described above. The washing buffer is thenremoved by centrifugation. The cells are then suspended in the polymersolution and incubated as described above. The polymer solution may beremoved by centrifugation. The cells are then optionally washed againwith a physiological buffer as described above to remove residualpolymer.

EXAMPLES

All starting materials, solvents, and reagents were purchased fromcommercial sources and used without further purification. Molecularweights are weight average and were determined by aqueous gel permeationchromatography (GPC) using polyethlyene glycol standards. Chemicalstructures were confirmed by ¹H and ¹³C NMR spectra measured at roomtemperature on a 400 MHz (100 MHz for ¹³C) instrument in D₂O or DMSO-d₆.

Examples 1-3

A polymer 4 comprising a recurring unit of the formula (VIII) isprepared by amidating a diamine and a glutamic amino acid derivativeaccording to the reaction scheme illustrated in FIG. 1 as follows: Asolution of glutamic amino acid derivative 2 (1.5 g, 3.2 mmol) inacetone (25 mL) is added into a solution of diamine 1 (0.87 g, 3.2 mmol)in acetone (10 mL) at room temperature with stirring. White precipitateforms after a few minutes. Stirring is continued for about 15 minutes.The solution is decanted and the residue is washed with additionalacetone. The resulting polymer 4 (0.87 g, 1.67 mmol) is obtained afterdrying the residue under high vacuum. The weight average molecularweight of the polymer 4 is about 45,000 daltons with a polydispersity ofabout 1.48. Similar reactions carried out in dichloromethane (DCM) anddimethylformamide (DMF) also produce polymer 4, in these cases havingweight average molecular weights of about 45,000 daltons (PDI 1.42) andabout 58,000 daltons (PDI 1.37), respectively.

Example 4

A polymer 5 comprising a recurring unit of the formula (IX) is preparedby amidating a diamine and a glutamic amino acid derivative according tothe reaction scheme illustrated in FIG. 1 in a manner similar to thatdescribed in Examples 1-3 using acetone as a solvent, except thatglutamic amino acid derivative 3 is used in place of 2 as illustrated inFIG. 1. The weight average molecular weight of the resulting polymer 5is about 10,000-12,000 daltons.

Example 5

A polymer 6 that comprises a recurring unit of the formula (VII) isprepared by a process that comprises removing a CBZ protecting groupfrom the polymer 4 (prepared as described in Examples 1-3) thatcomprises a recurring unit of the formula (VIII) via palladium/carboncatalytic hydrogenation according to the scheme illustrated in FIG. 1 asfollows: CAUTION!! Pd/C is highly flammable when flammable solvents arenear. This procedure should be conducted under an argon or nitrogenatmosphere. A sample of the polymer 4 (4.0 g) is added into a 500-mLflask equipped with a stirring bar. Pd/C (10%, 0.5 g) is added into theflask. The flask is purged with argon. Deoxygenated methanol (150 mL) isadded into the flask. Hydrogen gas (1 atm) is introduced and the mixtureis stirred under 1 atm hydrogen gas for 1 day. The insoluble residue isfiltered. The filtrate is concentrated by rotary evaporation to providea residue. The resulting polymer 6 (3.0 g) is obtained after drying theresidue under high vacuum.

Example 6

A polymer that comprises a recurring unit of the formula (I)(specifically, a polymer that comprises recurring units of the formulas(IV), (V) and (VI)) is prepared by a process that comprises amidatingthe polymer 6 (comprising a recurring unit of the formula (VII)) withNHS-dPEG₄-Biotin, glutaric anhydride, and oleic chloride according tothe scheme illustrated in FIG. 2 as follows: A sample of the polymer 6(105 mg) is dissolved in dimethylacetamide (DMA) (3 mL). Anhydrouspotassium carbonate (100 mg) is added into the mixture. NHS-dPEG₄-biotin(QuantaBiodesign, Ltd) (50 mg) is added into the mixture and stirred forabout 20 min. Glutaric anhydride (10 mg) is added and stirring iscontinued for about 2 hours. Oleic chloride (95 μL) is added andstirring is continued for about 5 min. The reaction is quenched withwater (5 mL) and acetone (5 mL) is added to induce precipitation. Theresidue is centrifuged and isolated. The residue is purified bysephadex-G25 gel filtration, and the product polymer IA comprising arecurring unit of the formula (I) is obtained in fractions afterfreeze-drying.

Example 7

A sample of polymer IA (prepared as in Example 6 above) was used in thisexample (see FIG. 3, polymer IA). The polymer was prepared at twodifferent concentrations of 10 μg/mL and 5 μg/mL in PBS.Streptavidin-Alexa 448 (Molecular Probes, Inc.) was diluted in 25 μg/mLin PBS. The streptavidin-green fluorescene Alexa was added to the cellsto monitor binding of the polymer to the cells. When Streptavidin bindsto the biotin moiety on the polymer, a green fluorescence is observed.

Lung cancer cells (A549 cells) were seeded in 6-well plates overnightwith approximately 50% confluency. Cell morphology was checked to insureno detachment from the plates. Buffers were removed from the cells andthe cells were washed twice with 2 mL PBS. PBS was used as the negativecontrol in one well. The other wells received 1 mL of the polymer IA ofFIG. 3 per well. The cells were incubated with the polymer solutions for15 min. The solution containing the polymer was removed. The cells werewashed again twice with PBS, 2 mL. Streptavidin-Alexa 488 solution wasadded (1 mL). The coated cells and negative control were incubated withthe streptavidin solution for 30 min. Convenient incubation times may befrom 2 min. to 1 hour. The solution containing the streptavidin wasremoved from the cells. The cells were washed again twice in 2 mL ofPBS. PBS (500 μL) was added and the cells were viewed in a fluorescencemicroscope. The results are shown in FIG. 4.

FIG. 4 shows that the cells treated with polymer IA as described aboveare strongly fluorescent due to the binding of thestreptavidin-fluorescene to the biotin moiety on the polymer. Incontrast, the control treatment shown on the right shows nofluorescence. Even though the fluorescent binding agent was added to thecontrol cells, there was no binding in the absence of the polymer. Thisexperiment demonstrates successful polymer coating of the cell.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

1. A polymer comprising at least one recurring unit represented by aformula selected from the group consisting of formulas (I), (II), and(III):

wherein n is 1 or 2; wherein x and y are each individually integers offrom about 1 to about 500; wherein Z is an optional linker groupcomprising from about zero to about 20 carbon atoms, from about zero toabout 5 oxygen atoms, from about zero to about five nitrogen atoms, fromabout zero to about 5 sulfur atoms, and from about zero to about fivephosphorous atoms; and wherein each W is individually selected from thegroup consisting of biotin, a fatty acid, a fluorescent dye, anantibody, a peptide, a targeting ligand, a polysaccharide, and anegatively charged group.
 2. The polymer of claim 1 in which the fattyacid comprises a moiety selected from the group consisting of oleic,stearyl, palmitic, linoleic, linolenoic, and cholesteryl.
 3. The polymerof claim 1 in which the negatively charged group is selected from thegroup consisting of C(═O)O⁻, SO₃ ⁻, or PO₄ ²⁻.
 4. The polymer of claim 1comprising at least one recurring unit selected from the groupconsisting of a recurring unit of the formula (IV), a recurring unit ofthe formula (V), and a recurring unit of the formula (VI):


5. The polymer of claim 4 comprising a recurring unit of the formula(IV), a recurring unit of the formula (V), and a recurring unit of theformula (VI).
 6. A polymer comprising a recurring unit of the formula(VII):


7. The polymer of claim 1 comprising a recurring unit of the formula(VIII):


8. The polymer of claim 1 comprising a recurring unit of the formula(IX):


9. A method of making the polymer of claim 1 that comprises a recurringunit of the formula (I)

comprising amidating a polymer that comprises a recurring unit of theformula (VII)

with a reagent selected from the group consisting of NHS-dPEG₄-Biotin,NHS-dPEG₄-peptide, glutaric anhydride, fatty acid chloride, andactivated ester NHS.
 10. A method of making the polymer of claim 1 thatcomprises a recurring unit of the formula (II), comprising amidating apolymer that comprises a recurring unit selected from the groupconsisting of glutamic acid recurring unit and glutamic acid saltrecurring unit, with an amino-peptide and a fatty acid amine.
 11. Amethod of making the polymer of claim 1 that comprises a recurring unitof the formula (III), comprising amidating a polymer that comprises arecurring unit selected from the group consisting of lysine recurringunit and lysine salt recurring unit, with a reagent selected from thegroup consisting of NHS-dPEG₄-Biotin, NHS-dPEG₄-peptide, glutaricanhydride, fatty acid chloride, and activated ester NHS.
 12. A method ofmaking the polymer of claim 4 that comprises a recurring unit of theformula (IV), comprising amidating a polymer that comprises a recurringunit of the formula (VII) with oleic chloride.


13. A method of making the polymer of claim 4 that comprises a recurringunit of the formula (V), comprising amidating a polymer that comprises arecurring unit of the formula (VII) with glutaric anhydride andpotassium carbonate


14. A method of making the polymer of claim 4 that comprises a recurringunit of the formula (VI), comprising amidating a polymer that comprisesa recurring unit of the formula (VII) with NHS-dPEG₄-Biotin


15. A method of making the polymer of claim 6, comprising removing aprotecting group from a polymer that comprises a recurring unit of theformula (VIII) via palladium/carbon catalytic hydrogenation


16. A method of making the polymer of claim 6, comprising treating apolymer that comprises a recurring unit of the formula (IX) with 20%piperidine


17. A method of making the polymer of claim 7, comprising amidating adiamine and a glutamic amino acid derivative, wherein the diamine isrepresented by the formula:

and wherein the glutamic amino acid derivative is represented by theformula:


18. A method of making the polymer of claim 8, comprising amidating adiamine and a glutamic amino acid derivative, wherein the diamine isrepresented by the formula:

and wherein the glutamic amino acid derivative is represented by theformula:


19. A polymer-coated cell comprising a living cell and a polymercomprising at least one recurring unit represented by a formula selectedfrom the group consisting of formulas (I), (II), and (III):

wherein n is 1 or 2; wherein x and y are each individually integers offrom about 1 to about 500; wherein Z is an optional linker groupcomprising from about zero to about 20 carbon atoms, from about zero toabout 5 oxygen atoms, from about zero to about five nitrogen atoms, fromabout zero to about 5 sulfur atoms, and from about zero to about fivephosphorous atoms; and wherein each W is individually selected from thegroup consisting of biotin, a fatty acid, a fluorescent dye, anantibody, a peptide, a targeting ligand, a polysaccharide, and anegatively charged group, the polymer being non-covalently attached toat least a portion of the exterior of the living cell.
 20. Thepolymer-coated cell of claim 19 in which the living cell is selectedfrom the group consisting of epithelial cell, endothelial cell,progenitor cell, mature stem cell, and embryonic stem cell.
 21. Thepolymer-coated cell of claim 19, wherein the polymer comprises at leastone recurring unit selected from the group consisting of a recurringunit of the formula (IV), a recurring unit of the formula (V), and arecurring unit of the formula (VI):


22. The polymer-coated cell of claim 19 wherein W is a targeting ligand.23. The polymer-coated cell of claim 22, wherein the targeting ligand isselected from the group consisting of transferrin and epidermal growthfactor.
 24. The polymer-coated cell of claim 19, wherein the antibody isa receptor that is overexpressed in cancer cells.
 25. The polymer-coatedcell of claim 19 in which the fatty acid comprises a moiety selectedfrom the group consisting of oleic, stearyl, palmitic, linoleic,linolenoic, and cholesteryl.
 26. The polymer-coated cell of claim 19 inwhich the negatively charged group is selected from the group consistingof C(═O)O⁻, SO₃ ⁻, or PO₄ ²⁻.
 27. The polymer-coated cell of claim 19,wherein the living cell is a mammalian cell.
 28. The polymer-coated cellof claim 19, wherein the living cell is a human cell.
 29. A method forcoating a living cell, comprising intermixing the living cell with apolymer comprising at least one recurring unit represented by a formulaselected from the group consisting of formulas (I), (II), and (III):

wherein n is 1 or 2; wherein x and y are each individually integers offrom about 1 to about 500; wherein Z is an optional linker groupcomprising from about zero to about 20 carbon atoms, from about zero toabout 5 oxygen atoms, from about zero to about five nitrogen atoms, fromabout zero to about 5 sulfur atoms, and from about zero to about fivephosphorous atoms; and wherein each W is individually selected from thegroup consisting of biotin, a fatty acid, a fluorescent dye, anantibody, a peptide, a targeting ligand, a polysaccharide, and anegatively charged group, wherein the polymer is intermixed with theliving cell in an amount effective to at least partially coat theexterior of the living cell.
 30. The method of claim 29, wherein thepolymer comprises at least one recurring unit selected from the groupconsisting of a recurring unit of the formula (IV), a recurring unit ofthe formula (V), and a recurring unit of the formula (VI):


31. The method of claim 29 in which the living cell is selected from thegroup consisting of epithelial cell, endothelial cell, progenitor cell,mature stem cell, and embryonic stem cell.
 32. The method of claim 29 inwhich the fatty acid comprises a moiety selected from the groupconsisting of oleic, stearyl, palmitic, linoleic, linolenoic, andcholesteryl.
 33. The method of claim 29 in which the negatively chargedgroup is selected from the group consisting of C(═O)O⁻, SO₃ ⁻, or PO₄²⁻.
 34. The method of claim 29, wherein the living cell is a mammaliancell.
 35. The method of claim 29, wherein the living cell is a humancell.